C3H/Sm mice have lost the exogenous milk-borne mammary tumor virus characteristic of the C3H strain and have a very low (1.5%) incidence of spontaneous mammary tumors. They are highly susceptible to mammary carcinogenesis by either chemical carcinogens or infection with the milk-borne virus. We have analyzed the MMTV proviral DNA content of normal tissues and of spontaneous, virus-induced, and chemically-induced mammary tumors by restriction endonuclease digestion and Southern blot analysis. While the results clearly show additional MMTV sequences in the virus-induced tumor not present in liver DNA, none of the spontaneous or chemically-induced tumors can be shown to contain newly acquired either exogenous or amplified endogenous MMTV sequences. Examination of the susceptibility of restriction sites to isoschizmers of the restriction endonucleases sensitive to the presence of methyl groups provided evidence that hypomethylation of some of the MMTV proviral sequences occur in the spontaneous and chemically induced C3H/StWi tumors. These sites are hypermethylated in normal C3H/StWi tissues suggesting that these genes are actively transcribed in the mammary tumors.